Aberrant DNA methylation is linked to a growing number of human diseases, such as myelodysplastic syndrome, acute myeloid leukemia, almost all solid tumor cancers, and a number of genetic syndromes such as Prader Willi Syndrome (PWS). There has been moderate success of treatments of myelodysplastic syndromes with the FDA approved demethylating nucleoside analogs, including azacitidine and decitabine. Unfortunately, these agents are also highly toxic by virtue of acting indiscriminately on the entire genome, thus bringing about the numerous side effects. Here we propose to develop novel tools and protocols for targeted gene-specific alteration of genomic methylation. These tools and protocols will not only be applicative in research, but, most importantly, could eventually ground the bases for novel therapeutic agents in cancer and other diseases. This proposal builds on our recent discoveries: (1) RNA deep sequencing (RNA-seq) analysis on RNAs immunoprecipitated with DNMT1 antibody, revealed ~6,000 transcripts interacting with DNMT1, suggestive of global involvement of transcription in the establishment and maintenance of cell type-specific DNA methylation patterns; (2) among these transcripts, the CEBPA gene locus noncoding RNA, chosen as a model for this study, was shown to be directly involved in inhibition of DNA methylation by forming complexes with DNA methyltransferase (DNMT1); and (3) expression of this RNA in cells in which CEBPA was not expressed resulted in promoter demethylation and activation of gene expression in a gene selective manner. These three major findings prompted us to propose the development of targeted gene-specific demethylation agent(s). Introduction of this novel gene-specific demethylating approach will lead to new treatments with great advantages over existing 5-aza-cytidine-based protocols. These advantages will include: a) high gene specificity; b) lower cytotoxicity; and c) absence of drug based side effects. These technologies will add to our ability to understand the basis of cancer development and progression, and form the basis of potentially novel therapeutic approaches.